Project Summary ? Project 2 This project has three aims addressing new aspects of transcription regulation. The first aim investigates the processes responsible for variation among cell states, such as those found in tumors, with a focus on the role of microRNAs (miRNAs). Data from single cell RNA sequencing provides new windows into these processes. In a cell population some genes vary in mRNA expression while others are uniform. Networks of co-varying genes define a range of possible transitional cell states. Elucidation of the structure of these networks will provide insights into the heterogeneity of cancers. The relationship between miRNA activity in embryonic cells and networks of pluripotent genes will be investigated in Aim 1, Definition of biological variation in gene networks due to microRNAs, with future focus on collaborations with the Jacks and Lees labs. The second aim investigates mechanisms underlying transcriptional variation. A liquid-liquid phase or, more specifically, gel-sol phase transition model is proposed as central to interactions between enhancers and promoters. This quantitative model predicts highly cooperative transitions for changes in the valences of interactions between macromolecules. The model is motivated by the high sensitivity of super-enhancers to inhibitors of general transcription factors. These inhibitors are now being tested in treatment of cancer. The relationship between super-enhancers and miRNA expression provides an experimental context to investigate the model, particularly the activity of super-enhancers in the efficient processing of primary miRNA and how human cancers with poor survival gain super-enhancers at genes for oncogenic miRNAs. Investigation in Aim 2, Regulation of transcription and RNA processing by super-enhancers and gel-sol phase transition biology, may enable identification of new cancer therapy targets. The third aim is a collaboration with Jackie Lees' lab analyzing the role of inhibition of the arginine dimethylase PRMT5. Lees' lab has found that glioblastoma cells vary dramatically in sensitivity to a small-molecule inhibitor of PRMT5. Establishing the bases of this differential sensitivity could allow identification of patients who would therapeutically benefit from PRMT5 inhibition. PRMT5 dimethylates residues in Sm proteins that comprise the snRNPs critical for spliceosome function. Analysis of RNA-seq data following inhibition of PRMT5 revealed a dramatic increase in ?detained? introns for a subset of genes and a decrease in the corresponding mRNAs. This will be investigated in Aim 3, Tumor sensitivity to PRMT5 inhibition through splicing regulation and snRNP.